The biosynthesis of the opioid pentapeptides enkephalins is under investigation. The primary enkephalin precursor, called preproenkephalin, was synthesized and studied by cell-free translation of messenger RNA from brain and adrenal medulla. The molecular weight of bovine, rat, and guinea pig preproenkephalin is 30,000. The bovine precursor was shown to contain both methionine- and leucine-enkephalin. Preproenkephalin and its biosynthetic intermediates were detected and quantitated by immunoprecipitation with anti-Met-enkephalin(ArgPhe) serum, followed by gel electrophoresis. Preproenkephalin was shown to be cotranslationally converted by microsomes to an apparently unglycosylated protein of Mr 28,000, called proenkephalin. Bovine preproenkephalain mRNA possesses 1450 plus or equal to 200 nucleotides. Monoclonal antibodies have been prepared against Met-enkephalin and Met-enkephalin(ArgPhe). A rat brain cDNA library has been prepared by recombinant DNA techniques for isolation of rat preproenkephalin clones and another clones of interest. The regulation of enkephalin gene expression is under investigation. In a related project, corticotropin-releasing factor (CFR) was shown to stimulate the release of beta-endorphin and corticotropin from clonal AtT-20 mouse pituitary tumor cells, as well as to stimulate adenylate cyclase of these cells.